Field of the Invention
The present invention concerns the repeated mechanical movements of swelling and contraction of bile canaliculi lumen making possible the canalicular lumen clearing, and the relationship with the Rho-kinase pathway and myosin light chain phosphorylation for molecular regulation of these movements. The invention also extends to cholestatic diseases with evidence of disorders in these movements associated with alteration of the Rho-kinase pathway activity and permeability of parajunctional spaces conditioning the lumen clearing. The invention is also concerned with methods for detection of these alterations and using derivatives for screening candidate compounds for susceptibility to induce canalicular functional activity, and for detecting diseases associated with biliary flow dysfunctions.
Description of Related Art
Cholestasis is a condition where bile cannot flow from the liver to the duodenum. A key functional parameter for good bile flow from hepatocytes is contraction of the bile canaliculi. Acute and chronic cholestasis results from dysfunction of the normal mechanisms of bile formation. Several forms of cholestatic disease can be produced by drugs. For example, chlorpromazine (CPZ) is known to induce intra-hepatic cholestasis in vivo. The effect was also described in human HepaRG® cells in vitro, with evidence of occurrence of bile canaliculi constriction. Several cases of cholestatic side effects have been reported with new drugs during the last few years. Therefore, the urgent challenge today is to increase the ability to improve the safety of therapeutics by better predicting these drug-induced adverse effects. For many concerned drugs the proposed explanation has mainly involved alteration of the hepatobiliary transport system, in particular reduction of available bile salt export pump molecules. A role for oxidative stress as a primary causal agent and/or an aggravating factor has also been supported by research. Concomitantly, other mechanisms such as cytoskeletal modifications and disruption of cell-to-cell junctions, all known to participate to cell polarity could be involved in altered bile salt flow in cholestasis. However, molecular mechanisms controlling this cascade of events in hepatocytes are poorly understood. FIG. 1 shows the hepatic lobule organization in liver tissue. FIG. 2 shows the junctional complex around the canalicular lumen, and the organization of a tight junction and localization of junction proteins.
Models and methods for detecting and characterizing drug-associated cholestatic disorders have been attempted. One problem relates to finding suitable hepatic models for in vitro studies. Hepatocyte polarization with bile canalicular formation is a complex mechanism which includes cytoskeletal, tight junctional and intracellular trafficking components. Limited availability of human fresh cells has led to use human cell lines. Generally permanent hepatic cell lines fail to form typical networks of bile canaliculi as in vivo and consequently, knowledge on the signaling pathways involved in bile canalicular lumen constriction and enlargement remains poor. Previous studies used WIFB9 or human HepG2 cells to describe the cell polarity and an interesting demonstration of the major role exerted by extracellular matrix signaling onto the canalicular formation using multilayered hepatic cords experimentally built with HepG2 cells, has been reported. However, these cells express only few functions characterizing the mature hepatocyte detoxification metabolism, including transport function. In contrast, recent research has indicated that differentiated human HepaRG cells which express phases 1 and 2 drug metabolizing enzymes and transporters, and form bile canaliculi structures, can be used to mimic features of intrahepatic cholestasis induced by CPZ treatment and to characterize the mechanisms involved in the initiation of the lesions. Bile canaliculi constriction and H2O2 production were mainly evidenced (Antherieu S. et al., 2013).
Methods have been established for analyzing cholestatic disorders. The general assumption up to now was that the major disorder expected to occur in cholestatic disorders was alteration of the hepatobiliary transport system associated with reduction of the number of available bile salt export pump molecules due to inhibition or competition between candidate compounds and bile salts. Thus, the general strategy for detection exclusively focused on:
i)—a study on levels of transporters expression and search for drug binding onto target transporters;
ii)—evidence and calculation of drug efflux alteration mainly using the radiolabelled bile salt precursor taurocholate as referent marker candidate (See e.g., U.S. Pat. No. 7,604,934).
This test consists in defining the bile efflux index using the following equation:
  BEI  =                                                                        Accumulation                ⁢                                                                  ⁢                                  Plus                  ⁡                                      (                    +                    )                                                  ⁢                Buffer                            -                                                                          Accumulation              ⁢                                                          ⁢                              Minus                ⁡                                  (                  -                  )                                            ⁢              Buffer                                                            AccumulationPlus          ⁡                      (            +            )                          ⁢        Buffer              ×    100  Total accumulation of radiolabelled taurocholate determined in HBSS Plus (+) buffer representing the total mass of compound taken up and excreted whereas the total mass of analyte inside the cells at the end of incubation is determined in Minus (−) buffer. (+) buffer is a Ca++ buffer and (−) buffer is Ca++-free buffer.
However, such assays have drawbacks. The main limitations of such assays are that: i)—it is designed primarily for studying biliary efflux of the candidate compounds and not necessarily the bile flow out the canaliculi; ii) it considers only the flow from the hepatocytes toward the bile canaliculi, and does not take into account the emptying of that bile canaliculi; iii)—Transporters, principally BSEP, are considered as the main factors involved in the modulation of biliary excretion; and iv)—it does not consider dynamic events of contraction/relaxation associated with efflux and clearing. Thus, it fails to highlight some important biological effects. For instance, there is no consideration of possible modification of the canalicular lumen (size) and analysis of mechanical activity (contractile movements) of bile canaliculi conditioning canalicular lumen clearing; v)—Index calculation is an endpoint; vi)—another limitation is the use of human primary cultures which have a limited lifetime even when they are used under sandwich conditions. In addition, reproducibility is always questionable due to known inter-individual variability.